Product 12/12
Rating: 7.1/10 (8 votes)
Catalog #: Q74104
The RNeasy Mini Kit allows efficient purification of total RNA from very small (see figure "RT-PCR of RNA from ≥100 Cells") amounts of starting material, up to 30 mg tissue or 1 x 107 cells . Total RNA is easily purified from animal cells or tissues, Gram-positive or Gram-negative bacteria,* or yeast† (See table "Total RNA Yields Obtained with RNeasy Mini Kits" and figure "High-Quality RNA from a Variety of Samples"). Protocols are also included for isolation of cytoplasmic RNA ‡ from eukaryotic cells as well as for cleanup of partially purified RNA, in vitro transcripts, and RNA from enzymatic reactions. A variety of special application protocols, including a protocol for vacuum processing of RNeasy mini columns, are also available. QIAshredder Homogenizers for convenient cell-lysate homogenization are available as an accessory.
RNeasy technology simplifies total RNA isolation by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-gel–membrane purification. There is no need for centrifugation through CsCl cushions or precipitation with LiCl or alcohol. RNeasy sample preparation technology is fully licensed, allowing RNeasy purified nucleic acids to be used in any molecular assay or other downstream application without risk of patent infringement.
RNeasy Kits provide the highest-quality RNA with minimum copurification of DNA. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using the RNase-Free DNase Set for convenient on-column DNase treatment during the RNeasy procedure.
* Lysozyme (required for bacterial samples) is not provided. † Lyticase, zymolase, or glass beads (required for yeast samples) are not provided. ‡ Additional buffer required. The buffer composition is provided in the protocols.
High-Quality RNA from a Variety of Samples
Formaldehyde agarose gel and northern blot of total RNA purified with the RNeasy Maxi Kit. 10 µg of total RNA isolated from each source was loaded per lane. All tissues were from mouse. Yeast: Saccharomyces cerevisiae; E. coli: HB101. 32P-labeled probes recognized (G) GAPDH; (E) translation elongation factor EF-1α ; and (O) outer membrane protein A sequences. (E and O were kindly provided by P. Philippsen, University of Basel, Switzerland and U. Henning, Max Planck Institute of Biology, Tübingen, Germany, respectively.) B. subtilis was not probed. M: 0.24–9.5 kb RNA ladder. 7.5 kb band (indicated) in embryo, Huh7, and HeLa cell lanes is a nuclear precursor RNA.
RT-PCR of RNA from >100 Cells
RT-PCR of total RNA isolated with the RNeasy Mini Kit from the indicated numbers of HeLa cells. 10 µl (1/5) of eluate was digested with RNase-free DNase and reverse transcribed with oligo-dT primer. 2.5 µl (1/20) of the cDNA mix was used in 50 µl PCR. A 452 bp fragment of GAPDH was amplified. C–: negative control; C+: positive control; M: 100 bp ladder.
Total RNA Yields Obtained with RNeasy Mini Kits
Amounts can vary due to developmental stage, species, growth conditions used, etc. Since the RNeasy procedure enriches for RNA species >200 nt, RNA yield does not include 5S rRNA, tRNAs, or other low-molecular-weight RNAs. * Using the specialized protocol for heart, muscle, and skin tissue, included in handbook. Proteinase K (required for this protocol) is not provided. † Higher yields can be obtained from stabilized bacteria using RNeasy Protect Bacteria Kits.
Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica-gel membrane. RNA binds, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in water.
RNA purified with RNeasy technology has A260/A280 ratios of 1.9–2.1* and is ideal for use in all applications. Downstream applications include:
